Lung surfactant protein D (SP-D) is involved in pulmonary surfactant homeostasis and is one of the collectins belonging to a family of oligomeric proteins consisting of a collagen region linked to a C-type lectin domain via an α-helical neck region. The structural subunits of SP-D are homotrimers of these chains, and the subunits are themselves oligomerized into cross-like tetramers and higher oligomers (3). SP-D binds to oligosaccharides on the surface of a variety of pathogenic microorganisms and induces aggregation. It initiates several effector mechanisms including the recruitment of inflammatory cells to destroy the pathogens. SP-D enhances the binding of influenza A virus to neutrophil granulocytes and promotes the neutrophil respiratory burst in response to the virus. It binds directly to alveolar macrophages in the absence of microbial ligands, and generates oxygen radicals (3, 4, 5). In addition to its role in antimicrobial defense, SP-D also seems to have an immunomodulatory function, inhibiting T lymphocyte proliferation and IL-2 production as well as inhibiting specific IgE binding to allergens and blocking allergen-induced histamine release from human basophils, incorporating the properties of modulating lung immune response in the lung allergic disease condition (6, 7, 8). Owing to the above properties, SP-D was shown to offer protection in allergic murine models of pulmonary hypersensitivity to fungal (Aspergillus fumigatus) and Derp allergens with significant reduction in antigen-specific IgE levels, blood eosinophilia and pulmonary cellular infiltration (9, 10).
Erpenbeck (2006) also showed inhibition of allergen-induced early airway response, reduced airway hyperresponsiveness and reduced eosinophilia in bronchoalveolar lavage and lung tissues, on treatment with SP-D, also suggesting its role in modulating lung immune response in the pulmonary allergic disease condition (11). Palaniyar et al, showed that SP-D increases clearance of apoptotic pulmonary macrophages (12). Mahajan et al, 2006, showed that SP-D interacts with eosinophils and increased clearance of apoptotic eosinophils by macrophage cell line and they also showed that SP-D increases survival and decreases apoptosis in eosinophils from healthy human subjects (13). This observation is exactly opposite to the subject matter of this patent where SP-D decreases survival and increases/induces apoptosis in eosinophils from patients of hypereosinophilia and cytokine activated eosinophils. Activated eosinophils are secreting inflammatory cytokines and mediators and contribute to pathogenesis. Apoptotic eosinophils are eosinophils which are undergoing a programmed cell death. SP-D induces apoptosis in activated eosinophils only.
A recent study in December 2006 has appeared on SP-D interaction with human eosinophils in the journal, Clinical and Experimental Allergy. The study also showed direct interaction of SP-D with human eosinophils isolated from healthy normals through its CRD that resulted in inhibition of eotaxin triggered chemotaxis and ECP-degranulation stimulated by Ca2+ ionophore in the eosinophils (14). This study did not evaluate effect of SP-D on eosinophils from patients or activated eosinophils and did not evaluate apoptosis.
Novelty of the present invention is in inducing increased apoptosis in eosinophils from patients and activated eosinophils in presence of SP-D. Persistence of apoptotic eosinophils leads to increased tissue damage in eosinophilia hence their clearance is very essential. Eosinophil infiltration into the tissue is caused due to eosionphil activation caused by allergens or any other idiopathic mediator.
A few compounds have been shown to help in induction and clearance of apoptotic eosinophils, however, this is the first report of SP-D involvement in induction of apoptosis in eosinophils derived from patients and activated eosinophils.
SP-D has also been indicated to be useful for the prevention and diagnosis of pulmonary emphysema (U.S. Pat. No. 6,838,428) (15). Emphysema is a type of chronic obstructive pulmonary disease (COPD) involving damage to the air sacs (alveoli) in the lungs. As a result, your body does not get the oxygen it needs. Emphysema makes it hard to catch breath along with a chronic cough and trouble in breathing during exercise.
Clark et al, have filed a patent on recombinant surfactant protein D compositions and methods of use thereof (United States Patent 20040259201) wherein they describe an rSPD(n/CRD) polypeptide, fragment, homologue, variant or derivative thereof for use in a method of treatment or prophylaxis of a disease wherein said disease or disorder comprises an inflammatory disease selected from the group consisting of allergy and inflammatory lung disease, allergy to the house dust mite (Dermatophagoides sp), a fungus or fungal spores of Aspergillus fumigatus, microbial infection of the lung. They also claim a method of reducing airway hyperresponsiveness, serum IgE levels or eosinophilia in an individual, the method comprising administering to the individual an rSPD(n/CRD) polypeptide and a method of reducing alveolar macrophage number in an individual, the method comprising administering to the individual an rSPD(n/CRD) polypeptide that enhances the clearance of apoptotic alveolar macrophages, or enhances the clearance of necrotic alveolar macrophages, or both (16). The patent specifically mentions use of SP-D for reducing eosinophilia associated with airway hyperresponsiveness and this is not in conflict with our proposed claims of use of a formulation comprising of SP-D for inducing apoptosis in eosinophils derived from patients and activated eosinophils and reducing eosinophilia in eosinophil mediated diseases other than airway hyper-responsiveness.
Machiko and Whitsett (Patent: WO2007056195) have patented SP-D for prevention and treatment of lung infections and sepsis. Surfactant protein D (SP-D) is a member of the collectin family of collagenous lectin domain-containing proteins that is expressed in epithelial cells of the lung. Administration of SP-D protein or fragments thereof is useful for the prevention or treatment of sepsis or lung infection (17).
The invention WO03035683 by Lyster et al, describes a novel form of surfactant protein D (SP-D). It discloses a specific in vitro SP-D assay and a method to detect an increased risk for the development of atherosclerosis (18).
The earlier studies from our group and others proposed the inhibitory effect of SP-D on T cell proliferation and thereby reduction in Th2 type of cytokines namely IL-4, IL-5 and IL-13 are probably responsible for downregulation of eosinophilia in mice treated with native or recombinant SP-D (19, 20). We for the first time show that direct interaction of SP-D with activated eosinophils (eosinophils secreting ECP, MBP and other molecules resulting in desquamation of epithelium and resulting in tissue damage observed in eosinophil mediated diseases) resulted in induction and increase of apoptosis in them.
Eosinophils are primarily thought of as hematopoietic cells. Whereas eosinophils normally account for only 1-3% of circulating leukocytes their numbers greatly increase in the peripheral blood and tissues in a variety of human diseases. Any perturbations that results in systemic or local eosinophilia can have profound effects on a patient. Salient eosinophil-mediated disorders are eczema, eosinophilic cardiomyopathy, eosinophilic gastroenteritis, hypereosinophilic syndrome, graft versus host disease, chronic fibrosis, a parasitic inflammatory disorder, drug reaction, eosinophilic pneumonias, episodic angioedema with eosinophilia, inflammatory bowel disease, eosinophilic leukemia and/or food enteropathy and hypereosinophilia associated diseases that include infections, cirrhosis and cancer. Various diseases in which their numbers greatly increase in the peripheral blood and/or tissues and result in the histotoxic conditions include neuromuscular diseases with eosinophilia, hypereosinophilic leukemias, hypereosinophilc syndromes (rare hematological diseases), skin diseases like eosinophilia-Myalgia syndrome, eosinophilic fascitis, capillary leak syndromes (IL-2), Churg-Strauss syndrome, toxic oil syndrome, parasitosis, respiratory diseases including chronic sinusitis, etc and few of these are described below (1, 2).
Neuromuscular diseases with eosinophilia involve a number of disorders associated with variable degrees of muscle, peripheral nerve, and connective tissue involvement. These include diffuse fasciitis or Shulman syndrome, eosinophilic myositis and eosinophilic polymyositis (1, 2, 21).
The hypereosinophilic syndrome (HES) encompasses clinical manifestations sharing 3 features (1) a peripheral eosinophil count of greater than 1.5×109/L for longer than 6 months; (2) evidence of organ involvement (cardiac and neurologic systems), excluding benign eosinophilia; and (3) an absence of other causes of eosinophilia, such as parasite infestation (most common cause of eosinophilia worldwide), allergy (most common cause of eosinophilia in the United States), malignancy, and collagen vascular disease (22).
Eosinophils in HES infiltrate multiple organs where they inflict tissue damage through the release of granule proteins. They also release proinflammatory cytokines (ie, interleukin 1 alpha, tumor necrosis factor-alpha, interleukin 6, interleukin 8, IL-3, IL-5, GM-CSF, macrophage inflammatory protein), which attract more eosinophils and other inflammatory cells to the area. The HES presentation can be acute (eg, stroke), as when cardiac and neurologic systems are involved, or, more commonly, HES has an insidious onset. In an NIH series, common symptoms included fatigue (26%), cough (24%), breathlessness (16%), muscle pains or angioedema (14%), and fever (12%). Mucocutaneous manifestations occur in 25-50% of patients. Cardiac involvement is the most common cause of mortality in HES. Some cases of HES turn into leukemia, and, as such, chromosomal abnormalities are at the root of some cases of HES.
Eosinophilic cellulitis (Wells syndrome) is an uncommon condition of unknown etiology. The presentation usually involves a mildly pruritic or tender cellulites like eruption with typical histologic features characterized by edema, flame figures, and a marked infiltrate of eosinophils in the dermis.
Eosinophilia-myalgia syndrome (EMS) is a disorder that causes inflammation by eosinophils in nerve, muscle, and connective tissue, which may include the fascia (as in Eosinophilic fascitis). EMS is an illness characterized by pruritus, cutaneous lesions, edema, sclerodermoid changes, and joint pain, in addition to dramatic myalgia and eosinophilia. This phase lasts weeks to months and is followed by a chronic phase characterized by sclerodermoid skin changes, neuropathy, neurocognitive deficits, continued myalgia, and muscle cramps. Other less common chronic manifestations involve the pulmonary, cardiac, and gastrointestinal systems (23).
Molecular events leading to Eosinophilia: Three cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), and interleukin 5 (IL-5) have been shown to promote the growth and the maturation of eosinophils and to induce the conversion of normal eosinophils to hypodense eosinophils. In particular, IL-5 activity is shown to be elevated in sera from patients with EMS. Therefore, in EMS, IL-5 may play a substantial role in the growth and the stimulation of eosinophils and in their conversion to the hypodense, cytotoxic form. Hypodense eosinophils are activated cells with increased survival and an increased capacity for cytotoxicity, and release inflammatory mediators, such as leukotrienes, histo-toxic cellular components, growth factors, cytokines, chemokines, bioactive lipid mediators, toxic oxygen metabolites, etc to the exterior and cause damage. The delayed eosinophil apoptosis makes a significant contribution to the increased tissue load of eosinophils.
In view of the role of eosinophils in diseases, the present invention is aimed at providing the use of formulation with SP-D in resolution of eosinophilic inflammations by inducing and/or increasing apoptosis in activated eosinophils towards the beneficial effects.
The present invention generally relates to a novel interaction of Surfactant protein-D with human eosinophils, wherein the molecule, induced and increased apoptosis of activated eosinophils derived from peripheral blood of individuals with hypereosinophilia.
Further in the present study, purified native human SP-D (native) has been used. Also, a recombinant homotrimer containing eight gly-x-y collagen repeat sequences, an alpha-helical, coiled-coil neck region, and the carbohydrate recognition domain of SP-D, a functionally active molecule expressed as inclusion bodies in Escherichia coli was used (24).